We discovered mcl-1, a member of the bcl-2 family, by screening for genes that increase in expression early in the differentiation of ML-1 human myeloblastic leukemia cells. Our further studies have revealed mcl-1 to be similar to bcl-2 in some characteristics and different in others: For example, mcl-1 can enhance cell viability under some apoptosis-inducing conditions, although it may have effects less pronounced than those of bcl-2. Similarly, mcl-1 is prominent in mitochondria and exhibits a distribution that overlaps with, but is not identical to, that of bcl-2. Finally, mcl-1 co-immunoprecipitates with a 22 KD protein, as does bcl-2. The mcl-1 protein exists as three species, visualized as three closely spaced bands on blots. A shift in the relative abundance of these bands occurs during ML-1 cell differentiation. In addition, certain of these bands are phosphorylated, phosphorylation occurring on serine. We now plan to determine how the expression and phosphorylation of the different species of mcl-1 relates to function. In Aim I, we will complete the characterization of the effects of mcl-1 on cell viability, determining whether mcl-1 can inhibit cell death under a broad range of conditions as can bcl-2. In Aim II, we will identify the 22 KD protein and further characterize its interaction with mcl-1. In Aim III, we will identify the structural differences that distinguish the different species of the mcl-1 protein; we will determine, for example, how phosphorylation contributes to the appearance of these different species. In Aim IV, we will determine how phosphorylation (and any other modifications of mcl-1 discovered in Aim III) influence both the effect of mcl-1 on cell viability and the interaction of mcl-1 with the 22 KD protein. In Aim V, we will determine when (e.g., how early) in the death process mcl-1 must be expressed in order to exert its effects on viability. Taken together, these studies on mcl-1 will contribute to an emerging understanding of how genes in the bcl-2 family are regulated, and how these genes, in turn, regulate cell viability versus death.